Metabolic Compartmentation in Cortical Synaptosomes: Influence of Glucose and Preferential Incorporation of Endogenous Glutamate into GABA

Metabolism of glutamine was determined under a variety of conditions to study compartmentation in cortical synaptosomes. The combined intracellular and extracellular amounts of [U-¹³C]GABA, [U-¹³C]glutamate and [U-¹³C]glutamine were the same in synaptosomes incubated with [U-¹³C]glutamine in the presence and absence of glucose. However, the concentration of these amino acids was decreased in the latter group, demonstrating the requirement for glucose to maintain the size of neurotransmitter pools. In hypoglycemic synaptosomes more [U-¹³C]glutamine was converted to [U-¹³C]aspartate, and less glutamate was re-synthesized from the tricarboxylic acid (TCA) cycle, suggesting use of the partial TCA cycle from -ketoglutarate to oxaloacetate for energy. Compartmentation was studied in synaptosomes incubated with glucose plus labeled and unlabeled glutamine and glutamate. Incubation with [U-¹³C]glutamine plus unlabeled glutamate gave rise to [U-¹³C]GABA but not labeled aspartate; however, incubation with [U-¹³C]glutamate plus unlabeled glutamine gave rise to [U-¹³C]aspartate, but not labeled GABA. Thus the endogenous glutamate formed via glutaminase in synaptic terminals is preferentially used for GABA synthesis, and is metabolized differently than glutamate taken up from the extracellular milieu.     

Metabolic Compartmentation in Living Cells: Structural Association of Aldolase

The glycolytic enzyme aldolase is concentrated in a domain around stress fibers in living Swiss 3T3 cells, but the mechanism by which aldolase is localized has not been revealed. We have recently identified a molecular binding site for F-actin on aldolase, and we hypothesized that this specific binding interaction, rather than a nonspecific mechanism, is responsible for localizing aldolasein vivo.In this report, we have used fluorescent analog cytochemistry of a site-directed mutant of aldolase to demonstrate that actin-binding activity localizes this molecule along stress fibers in quiescent cells and behind active ruffles in the leading edge of motile cells. The specific cytoskeletal association of aldolase could play a structural role in cytoplasm, and it may contribute to metabolic regulation, metabolic compartmentation, and/or cell motility. Functional duality may be a widespread feature among cytosolic enzymes.     

The rate of turnover of cortical GABA from [1-13C]glucose is reduced in rats treated with the GABA-transaminase inhibitor vigabatrin (γ-vinyl GABA)

Brain GABA levels rise and plateau following prolonged administration of the irreversible GABA-transaminase inhibitor vigabatrin (γ-vinylGABA). Recently it has been shown that increased GABA levels reduces GAD₆₇ protein, one of two major isoforms of glutamic acid decarboxylase (GAD). The effects of GABA elevation on GABA synthesis were assessed in vivo using¹H and¹³C-edited NMR spectroscopy. Rates of turnover of cortical glutamate and GABA from intravenously administered [1-¹³C]glucose were measured in α-chloralose anesthetized rats 24 hours after receiving vigabatrin (500 mg/kg, i.p.) and in non-treated controls. GABA concentration was increased 2-fold at 24 hours (from 1.3±0.4 to 2.7±0.9 μmol/g) and GABA-T activity was inhibited by 60%. Tricarboxylic acid cycle flux was not affected by vigabatrin treatment compared to non-treated rats (0.47±0.19 versus 0.52±0.18 μmol/g, respectively). GABA-C2 fractional enrichment (FE) measured in acid extracts rose more slowly in vigabatrin-treated compared to nontreated rats, reaching >90% of the glutamate FE after 3 hours. In contrast, GABA FE≥glutamate FE in non-treated rats. A metabolic model consisting of a single glutamate pool failed to account for the rapid labeling of GABA from glutamate. Metabolic modelling analysis based on two (non-communicating) glutamate pools revealed a ∼70% decrease in the rate of GABA synthesis following vigabatrin-treatment, from 0.14 (non-treated) to 0.04 μmol/g/min (vigabatrin-treated). These findings, in conjunction with the previously reported differential effects of elevated GABA on the GAD isoforms, suggests that GAD₆₇ may account for a major fraction of cortical GABA synthesis in the α-chloralose anesthetized rat brain in vivo. Special issue dedicated to Dr. Herman Bachelard.     

Compartmentation and release of exogenous GABA in sheep brain synaptosomes

Exogenous tritiated -aminobutiric acid ([³H]GABA) is retained in two compartments in sheep cortex synaptosomes, corresponding to cytoplasmic and vesicular spaces, assuming that freeze-thawing the synaptosomes loaded with [³H]GABA releases the cytoplasmic [³H]GABA (81±3.9%), and that subsequent solubilization of the synaptosomes with 1% sodium cholate releases the vesicular [³H]GABA (19±3.9%). Depolarization of synaptosomes with 40 mM K⁺ in a Na⁺-medium, in the absence of Ca²⁺, releases 20.3±2.7% of the [³H]GABA retained in the synaptosomes. The [³H]GABA released under these conditions comes predominantly from the cytoplasm. The presence of 1 mM Ca²⁺ during depolarization releases and additional 13% (a total of about 33.5±9.9%) of the releasable [³H]GABA, and the [³H]GABA release which is Ca²⁺-dependent also comes mostly from the cytoplasmic compartment. When choline replaces external Na⁺, the [³H]GABA release is absolutely Ca²⁺-dependent, and the [³H]GABA released also comes mostly from the cytoplasmic pool. Therefore, it appears that [³H]GABA taken up by synaptosomes is accumulated mostly in the cytoplasmic compartment from which it is released upon depolarization. The technique described permits distinguishing the effect of different factors on the two pools of accumulated [³H]GABA.     

Regional Metabolism of Met-Enkephalin and Cholecystokinin on Intact Rat Brain Slices: Characterization of Specific Peptidases

Abstract: The metabolism of Met-enkephalin and cholecystokinin (CCK) 8-(sulfated) by intact microslices was studied in rat brain regions. Incubation of brain slices with Met-enkephalin (400 µ M ) resulted in a linear rate of disappearance of parent peptide and appearance of metabolic fragments whose rate of accumulation was specific to brain region. The degradative rate (pmol/min/mg of protein) of Met-enkephalin was high in caudate-putamen (5,160 ± 120) and lower in nucleus accumbens (3,630 ± 110) and frontal cortex (3,180 ± 120). Inhibition of aminopeptidases decreased Met-enkephalin degradation (50–97% vs. control) in frontal cortex but was less effective in caudate-putamen (20–34%). Tyr-Gly-Gly and Phe-Met were recovered in caudate-putamen and nucleus accumbens, whereas negligible quantities of these fragments were recovered from frontal cortex. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11, decreased Met-enkephalin degradation in caudate-putamen (14%) but had no effect on that in frontal cortex. A cocktail of bestatin or leuhistin (inhibitors of aminopeptidases), phosphoramidon, and captopril (an inhibitor of angiotensin converting enzyme) protected Met-enkephalin from degradation (recovery >95%) in caudate-putamen. CCK 8-(sulfated) degradation on slices from caudate-putamen, nucleus accumbens, and frontal cortex was not altered by inhibitors of neutral endopeptidase 24.11, metalloendopeptidase 24.15, angiotensin converting enzyme, or thiol proteases. Inhibitors of either aminopeptidases or serine proteases produced small reductions (13–30%) in CCK degradation in each region. These data provide evidence for regional and structural specificity in terminating the actions of neuropeptides.     

Ethanol,not detectably metabolized in brain,significantly reduces brain metabolism,probably via action at specific GABA(A) receptors and has measureable metabolic effects at very low concentrations

Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we measured the effect of alcohol on metabolism of [3‐¹³C]pyruvate in the adult Guinea pig brain cortical tissue slice and compared the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2‐¹³C]ethanol. Analyses of metabolic profile clusters suggest that the significant reductions in metabolism induced by ethanol (10, 30 and 60 mM) are via action at neurotransmitter receptors, particularly α4β3δ receptors, whereas very low concentrations of ethanol may produce metabolic responses owing to release of GABA via GABA transporter 1 (GAT1) and the subsequent interaction of this GABA with local α5‐ or α1‐containing GABA(A)R. There was no measureable metabolism of [1,2‐¹³C]ethanol with no significant incorporation of ¹³C from [1,2‐¹³C]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabelled ethanol.

     

Compartmentation and flux characteristics of nitrate in spruce

The radiotracer¹³N was used to undertake compartmental analyses for NO ₃ ^– in intact non-mycorrhizal roots ofPicea glauca (Moench) Voss. seedlings. Three compartments were defined, with half-lives of exchange of 2.5 s, 20 s, and 7 min. These were identified as representing surface adsorption, apparent free space, and cytoplasm, respectively. Influx, efflux, and net flux as well as cytoplasmic and apparent-free-space nitrate concentrations were estimated for three different concentration regimes of external nitrate. After exposure to external NO ₃ ^– for 3 d, influx was calculated to be 0.09 mol·g^–1·h^–1 (at 10 M [NO ₃ ^– ]_o), 0.5mol·g^–1·h^–1 (at 100 M [NO _inf3 ^sup– ]_o), and 1.2 mol · g^–1· h^–1 (at 1.5 mM [NO ₃ ^– ]_o). Efflux increased with increasing [NO ₃ ^– ]_o, constituting 4% of influx at 10 M, 6% at 100 M, and 21% at 1.5 mM. Cytoplasmic [NO ₃ ^– ] was estimated to be 0.3 mM at 10 uM [NO ₃ ^– ]_o, 2mM at 100 M [NO ₃ ^– ]_o, and 4mM at 1.5 mM [NO ₃ ^– ]_o, while free-space [NO ₃ ^– ] was 16 M, 173 M, and 2.2 mM, respectively. A series of experiments was carried out to confirm the identity of the compartments resolved by efflux analysis. Pretreatment at high temperature or application of 2-chloro-ethanol, sodium dodecyl sulphate or hydrogen peroxide made it possible to distinguish the metabolic (cytoplasmic) phase from the remaining two (physical) phases. Likewise, varying [Pi] of the medium altered efflux and thereby [NO ₃ ^– ]_cyt, but did not affect [NO ₃ ^– ]_{free space}.Abbreviations and Symbols [NO ₃ ^– ]_cyt cytoplasmic NO ₃ ^– concentration - [NO ₃ ^– ]_{free space} apparent-free-space NO ₃ ^– concentration - [NO ₃ ^– ]_o concentration of NO ₃ ^– in the external solution - NO ₃ ^– flux - _co efflux from the cytoplasm - _oc influx to the cytoplasm - _net net flux - _xylem flux to the xylem - _red/vac combined flux to reduction and the vacuole The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia Campus for providing¹³NO ₃ ^– , Drs. R.D. Guy and S. Silim for providing plant material, and Dr. M.Y. Wang, Mr. J. Mehroke and Mr. P. Poon for assistance in experiments and for helpful discussions.     

Cerebral Ammonia Metabolism in Hyperammonemic Rats

The short-term metabolic fate of blood-borne [13N]ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a "freeze-blowing" technique it was shown that the overwhelming route for metabolism of blood-borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L-[amide-13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.     

Enzymes of Cerebral GABA Metabolism and Synaptosomal GABA Uptake in Acute Liver Failure in the Rabbit: Evidence for Decreased Cerebral GABA-Transaminase Activity

Abstract: Measurements of the activities of the two key enzymes in cerebral GABA metabolism—glutamate decarboxylase (GAD) and GABA-transaminase (GABA-T)—were performed in normal rabbits and in rabbits with hepatic encephalopathy due to galactosamine-induced liver failure. Furthermore the uptake of GABA by synaptosomes was studied. Hepatic encephalopathy was associated with a marked decrease in the activity of GAB A-T. This decrease in activity was already apparent in galactosamine-treated rabbits before the onset of hepatic encephalopathy. Sera and serum ultrafiltrates of rabbits with hepatic encephalopathy but not of normal rabbits or of rabbits with uremic encephalopathy were shown to inhibit GABA-T activity in vitro . Cerebral GAD activity and synaptosomal GABA uptake in rabbits with hepatic encephalopathy and in untreated animals were not different. These later findings indicate that hepatic encephalopathy is not associated with alterations of presynaptic GABA nerve terminals in the central nervous system. The demonstration of a decrease in cortical GABA-T activity provides indirect evidence for decreased GABA turnover in the brains of rabbits with hepatic encephalopathy and thus is compatible with augmented GABA-ergic inhibitory neurotransmission contributing to the neural inhibition of hepatic encephalopathy.     

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

Abstract: The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 m M [U-¹³C]glutamate 85% of the ¹³C metabolized was converted to glutamine. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 m M glutamate, ¹³C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [¹³C]lactate was essentially unchanged within the range of 0.2–0.5 m M glutamate, whereas the amount of [¹³C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 m M , suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 m M and astrocytes from both rat and mouse brain are consistent with these findings.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Synthesis of Cyclopropane Isosteres of the Antiepilepsy Drug Vigabatrin and Evaluation of their Inhibition of GABA Aminotransferase

The antiepilepsy drug vigabatrin (1; 4-aminohex-5-enoic acid; γ-vinyl GABA) is a mechanism-based inactivator of the pyridoxal 5'-phosphate (PLP)-dependent enzyme γ-aminobutyric acid aminotransferase (GABA-AT). Inactivation has been shown to proceed by two divergent mechanisms (Nanavati, S. M. and Silverman, R. B. (1991) J. Am. Chem. Soc. 113, 9341–9349), a Michael addition pathway (Scheme 2, pathway a) and an enamine pathway (Scheme 2, pathway b). Analogs of vigabatrin with a cyclopropyl or cyanocyclopropyl functionality in place of the vinyl group (2–5) were synthesized as potential inactivators of GABA-AT that can inactivate the enzyme only through a Michael addition pathway, but they were found to be only weak inhibitors of the enzyme.     

GABA concentrations in the striatum following repetitive cerebral ischemia

GABAergic neurons in the striatum are very sensitive to the effects of ischemia. The progressive decline in striatal GABA following transient forebrain ischemia in gerbils may be secondary to either a decreased production or an increase in reuptake mechanisms or both. The current experiment was designed to evaluate release of GABA by stimulation with K+ or inhibition of its uptake with nipecotic acid or their combination (K+ nipecotic) after repetitive forebrain ischemia in gerbils by in-vivo microdialysis on Days 1, 3, 5, and 14 following the insult. Infusion of nipecotic acid or potassium chloride, resulted in a significant increase in extracellular GABA. This response was significantly decreased in the post-ischemic animals. The synergistic effect of increased GABA concentrations by the infusion of nipecotic acid+potassium chloride seen in the controls was not evident in the post-ischemic animals. In conclusion, though there is a reduction in the extracellular GABA concentrations in the first week following an ischemic insult, restorative mechanisms are operative in the second week as seen by the increasing GABA concentrations.     

Mild reduction in the activity of the α-ketoglutarate dehydrogenase complex elevates GABA shunt and glycolysis

Diminished energy metabolism and reduced activity of brain α-ketoglutarate dehydrogenase complex (KGDHC) occur in a number of neurodegenerative diseases. The relation between diminished KGDHC activity and altered energy metabolism is unknown. The present study tested whether a reduction in the KGDHC activity would alter cellular metabolism by comparing metabolism of [U-¹³C]glucose in a human embryonic kidney cell line (E2k100) to one in which the KGDHC activity was about 70% of control (E2k67). After a 2 h incubation of the cells with [U-¹³C]glucose, the E2k67 cells showed a greater increase in ¹³C labeling of alanine compared with the E2k100 cells. This suggested an increase in glycolysis. Furthermore, an increase in labeled lactate after 12 h incubation supported the suggestion of an increased glycolysis in the E2k67 cells. Increased GABA shunt in the E2k67 cells was indicated by increased ¹³C labeling of GABA at both 2 and 12 h compared with the control cells. GABA concentration as determined by HPLC was also increased in the E2k67 cells compared with the control cells. However, the GABA shunt was not sufficient to normalize metabolism in the E2k67 cells compared with control at 2 or 12 h. However, by 24 h metabolism had normalized (i.e. labeling was similar in E2k67 and E2k100). Thus, the data are consistent with an enhanced glycolysis and GABA shunt in response to a mild reduction in KGDHC. Our findings indicate that a mild change in KGDHC activity can lead to large changes in metabolism. The changes may maintain normal energy metabolism but make the cells more vulnerable to perturbations such as occur with oxidants.     

葡萄果肉细胞脱落酸的区隔化

以处于不同发育时期的红富士葡萄 (VitisviniferaL .×VitislabruscaL .)果实为试材 ,采用胶体金免疫电镜定位技术对果肉细胞脱落酸 (ABA)的区隔化及其动态进行了研究。结果表明 :在果实发育前期 ,ABA主要存在于细胞核和胞质溶胶中 ;随着果实的进一步发育成熟 ,ABA转为主要分布在叶绿体和细胞核中。与处于第Ⅰ期、始熟期以及第Ⅲ期果实的果肉细胞ABA含量相比 ,处于第Ⅱ期的前期果实的果肉细胞ABA含量最低。这些事实说明在果实发育成熟的任一时期 ,果肉细胞ABA均呈区隔化分布 ,其在不同区隔之间的浓度差异随着果实的发育成熟而发生变化 ,即发生了再分配     

金针菇酶促褐变的区域分布及调控

金针菇的多酚氧化酶(PPO)、过氧化物酶(POD)和过氧化氢酶(CAT)活性在全株中呈现区域化分布。菌盖的酶活性最低,菌柄上部酶活性稍高,菌柄中部较高,菌柄下部活性最高。用硫脲、亚硫酸盐、CaCl₂、低CO₂、低温等方法处理金针菇,对以上三种酶的活性均有抑制作用。高0₂、H₂O₂和温度的提高均促进三种酶活性。高N₂(减压后充N₂)不能抑制三种酶活性。     

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution ¹³C NMR. The oxidation of [1,2,3-¹³C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS^?) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The ¹³C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure(TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the ¹³C spectra of cells presented with [3-¹³C] propionate or [2-¹³C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.